The invention relates to a process for the preparation of L-menthol by enantioselective enzymatic cleavage of D,L-menthyl derivatives.
Process for the synthetic preparation of menthol are generally known (Common Fragrance and Flavor Materials; Bauer, K., Garbe, D. and Surburg, H., Verlag V C H, Weinheim, 1990, 2nd edition, pp. 44-46). If the products obtained are racemic mixtures, they are markedly inferior in taste and odor to the naturally occurring L-menthol, for example from peppermint oil. Therefore, there is a great interest in separation processes for D,L-menthol.
The separation can be achieved, for example, using physical processes. Such processes include, for example, fractional crystallization of the salts of optically active amines with racemic methyl hydrogen phthalate or methyl hydrogen succinate. In addition, D- or L-menthol can be separated off from racemic menthol mixtures by esterifying the mixture with an optically active acid, for example menthoxyacetic acid, and separating the mixture of diastereomeric compounds by crystallization. The D- or L-menthol is obtained by saponification of the diastereomeric ester.
A further process used industrially (DE-A 2 109 456) for separating off optically pure D- and L-menthol from D,L-menthol mixtures proceeds via a carboxylic menthyl ester as intermediate. Preferably, the esters of benzoic acid or of hexahydrobenzoic acid, and in addition the esters of 4-methylbenzoic acid, of 3,5-dinitrobenzoic acid and of 4-ethoxybenzoic acid are used. The process is the selective crystallization of optical antipodes which are obtained in a purity so high that further processing can be carried out without further purification operations.
In addition, L-menthol can be isolated from D,L-menthol mixtures using enzymes or microorganisms.
It is also known that lipases hydrolyze esters in aqueous media and can have a high specificity and selectivity. In addition, in certain organic solvents, some lipases have the ability to catalyze the back-reaction, to synthesize esters from the corresponding acids and alcohols.
Various strategies have been employed to produce pure L-menthol from the racemic D,L-menthol mixture. Thus, for example, Tetrahedron Letters, 27, (1986) 29 discloses that the lipase of Candida cylindrarea preferentially releases L-menthol (ee: 70%) from a racemic menthyl laurate by hydrolysis in an aqueous medium. This enantioselective preference was also displayed in the esterification of racemic menthol with lauric acid, the L-menthyl laurate being formed with high enantiomeric purity (ee: 86%). In a non-aqueous medium, racemic menthol can be enantioselectively esterified with lauric acid using lipase, with, again, the L-menthyl laurate being formed preferentially (ee: 95%). This reaction is virtually complete after 10 hours. Transesterification of D,L-menthol with trilaurin or D,L-menthyl laurate with isobutanol proceeds with a similarly high enantioselectivity, but is extremely slow (reaction time: 15 days or more).
It is also known to carry out reactions under enzyme catalysis in nonaqueous media, if the substances are only poorly soluble in water. As an alternative to organic solvents, supercritical fluids, specifically supercritical carbon dioxide, may be used. Thus, this is also disclosed for racemate resolution of D,L-menthol by Chemie Ingenieur Technik, 69, (1986) 29, more precisely by the enantioselective transesterification of various acetates with racemic menthol. The best results are achieved with the enol ester isopropenyl acetate. Such esters have the benefit that after reaction is complete, the alcohol formed by the hydrolysis, in this case isopropenyl alcohol, immediately isomerizes to form the corresponding ketone and is therefore not available for any back-reaction. The enzymes studied are lipase AY from Candida rugosa, lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia), Novozyme 435 from Candida antarctica B, lipozyme IM 60 from Rhizomucor miehei and esterase EP 10 from Pseudomonas marginata. 
Esterase EP 10 can be obtained from recombinant E. coli strains which contain the gene for EP 10 esterase. Esterase EP 10 shows by far the highest enantioselectivities in the system. Novozyme 435, under the conditions selected, shows virtually no conversion in the transesterification using the various acetates.
The enantioselectivity of the lipase from Candida rugosa(lipase AY) towards racemic menthol may be significantly increased, according to the reports in Biotechnol. Prog. 11, (1995) 270 by targeted treatment of the lipase with nonionic surfactants. These studies clearly show that the effectivity of esterification of L-menthol with lauric acid in organic medium depends greatly on the enzyme. The lipase from Candida rugosa is significantly more effective in this reaction than the lipase from Rhizopus sp., Burkholderia cepacia, Pseudomonas sp., Mucor javanicus, Aspergillus niger and from pig pancreas. In addition, it is found that as a result of the treatment with nonionic surfactants, the effectivity of the lipase from Candida rugosa increases to about five fold.
Tetrahedron Letters 39, (1998) 4333 discloses that using microwave irradiation, in the case of pig pancreas lipase, leads to no change in reaction velocity or enantioselectivity in the esterification of racemic menthol with palmitic acid.
Lipases are also able to accept carboxylic anhydrides as acyl donor. Carboxylic anhydrides, as has already been mentioned in the case of the enol esters, have the advantage that acyl transfer is quasi-irreversible. According to Enzyme and Microbial Technology 18, (1996) 536, the lipase AY-30 from Candida rugosa is able to exercise a certain enantioselectivity in the reaction of racemic menthol with acetic anhydride, propionic anhydride and butyric anhydride. The best results with this enzyme are achieved with butyric anhydride after 48 hours in n-hexane as solvent (ee: 86% of the L-menthyl butyrate formed).
The enantioselectivity of the reaction is greatly dependent both on the lipase used and on the anhydride used. Thus, Microbiol. Biotechnol 43, (1995) 639, discloses that the lipase OF 360 from Candida rugosa and propionic anhydride gives a very high optical purity of the L-menthyl propionate formed (ee: 95%).
A further possible method of preparing L-menthol from D,L-menthol mixtures is to cleave racemic ester mixtures enantioselectively enzymatically. Thus, Dechema Biotechnol. Conf. (1989) 141 discloses reacting D,L-menthyl acetate with the lipase from Candida rugosa in a hydrolysis, the L-menthol released indicating a rather low enantioselectivity of the enzyme.
It is an object of the present invention to resolve a D,L-menthol suitable for industrial use, or derivatives thereof, with high absolute enantioselectivity, in order to obtain pure L-menthol or D-menthol or a pure L-menthyl ester or D-menthyl ester.
A process has been found for the preparation of D- or L-menthol and derivatives, characterized in that D,L-menthyl derivatives are enantioselectively enzymatically cleaved by lipases.
According to the inventive process, the enantiomers are surprisingly obtained at an enantiomeric excess (ee value) of greater than 99%, and a selectivity (E value) of  greater than 100.
D,L-Menthyl derivatives for the inventive process are, for example, compounds of the formula 
where
R denotes hydrogen, unbranched or branched C1-C20-alkyl, C3-C8-cycloalkyl, C6-C14-aryl, C7-C15-arylalkyl, C1-C20-alkoxy, C1-C20-alkylamino, where the above-mentioned hydrocarbon radicals can optionally be monosubstituted or polysubstituted with hydroxyl, formyl, oxy, C1-C6-alkoxy, carboxyl, mercapto, sulfo, amino, C1-C6-alkylamino or nitro or halogen, preferably chlorine.
Preferred D,L-menthyl derivatives are esters of D,L-menthol with aliphatic or aromatic carboxylic acids. For example, the following esters may be mentioned:
D,L-menthyl acetate, D,L-menthyl benzoate, D,L-menthyl isovalerate.
In particular, preference is given to D,L-menthyl benzoate.
The D,L-menthyl derivatives for the inventive process are known per se.
Usually, for the inventive process, lipases from Candida rugosa are used.
It is known that lipases can also be produced by recombinant DNA techniques (EP A 238 023). In these the lipase-coding gene is transferred from a selected strain by methods known to those skilled in the art to a receiving organism. This receiving organism produces the lipase.
In a most preferred embodiment, recombinant lipases which are immobilized on a support material are used. Suitable support materials are, for example, plastics such as polypropylene, polystyrene, polyvinyl chloride, polyurethane, polyacrylate, latex, nylon or Teflon, polysaccarides such as agarose or dextran, ion-exchange resins (both cationic and anionic), silicone polymers, for example siloxanes, or silicates, for example glass. Immobilization methods for enzymes are known to those skilled in the art (K. Mosbach, xe2x80x9cImmobilized Enzymesxe2x80x9d, Methods in Enzymology 44, Academic Press, New York, 1976) and comprise cross-linking, adsorption or covalent bonding to the support material.
Lipases from Candida rugosa are also commercially marketed, for example lipase AY (distributor: Amano, Nagoya, Japan).
Surprisingly, in a preferred form of the present invention it has been found that hydrolysis of D,L-menthyl benzoate using recombinant lipase from Candida rugosa (WO 99/14338) proceeds with very high enantioselectivity (E greater than 100) and an enantiomeric excess of (xe2x88x92)-menthol of  greater than 99.9%. This result has been confirmed by gas-chromatographic analysis, NMR spectroscopy and polarimetry.
The differing hydrolytic behavior of the two Candida rugosa lipases (commercial and recombinant) can be explained by the fact that commercial preparations can contain not only the desired enzyme, but a great number of isoenzymes having somewhat different properties. SDS-PAGE studies have found that the recombinant lipase used shows only one protein band (see WO 99/14338), while lipase AY shows a plurality of protein bands.
Customarily, the solvent used for the inventive process can be water, aqueous buffer and organic solvents. Organic solvents preferably used are hexane, cyclohexane, heptane, cycloheptane, toluene, dichloromethane, acetonitrile, dimethylformamide, dioxane, tetrahydrofuran or ethanol. The aqueous buffer preferably used is phosphate buffer or acetate buffer.
For the inventive process, generally, 1 to 10000 units (U) are used, preferably 10 to 1000 units (U) of the lipase, based on 0.01 mmol of the menthyl derivative.
The cleavage according to the inventive process is generally carried out in a temperature range from 0 to 90xc2x0 C., preferably from 20 to 60xc2x0 C.
The cleavage according to the inventive process is generally carried out in the pH range from 1 to 12, preferably at about pH 7.
The inventive process can be carried out, for example, as follows: In a first step the enzyme is produced in a fermenter in a similar manner to WO 99/14338 (see Example 1). In a second step, the resultant lipase is purified (see Example 1). In a third step the menthyl derivative is enzymatically cleaved (see Example 3).
The pure menthol enantiomers, thus prepared, comply with high analytical and sensory requirements.